Combination of Local and Systemic Immunomodulative Therapies for Enhanced Treatment of Cancer

ABSTRACT

A method for the treatment of cancer comprising administration of a therapeutically effective amount of an intralesional chemoablative pharmaceutical composition, or variant of said composition, in combination with a therapeutically effective amount of a systemic immunomodulatory anticancer agent. A further method for the treatment of cancer comprising administration of a therapeutically effective amount of an intralesional chemoablative pharmaceutical composition, or variant of said composition, in combination with a therapeutically effective amount of a systemic targeted anticancer agent. The present invention is further directed to pharmaceutical compositions for treatment of cancer. The intralesional chemoablative pharmaceutical composition can comprise an IL chemoablative agent comprising primarily a halogenated xanthene.

FIELD OF THE INVENTION

This invention relates to the fields of oncology and improvedtherapeutic regimens therefore.

BACKGROUND OF THE INVENTION

Pharmacologic approaches for treating cancer have traditionally reliedon the use of various single agent systemic therapies (monotherapies).An archetypical example is chemotherapy, which utilizes broadlycytotoxic drugs that target rapidly dividing cells, including alkylatingagents like dacarbazine (DTIC) or temozolomide (TMZ), or mitoticinhibitors like paclitaxel, to inhibit or kill the rapidly growing cellstypical of cancer. Tumors may not be completely responsive to suchmonotherapy, either due to their high collateral systemic toxicitynecessitating lower, even sub-therapeutic doses or development of tumorresistance that circumvents the activity of the monotherapy agent. Moreadvanced chemotherapy strategies have been developed that are predicatedon use of multiple agents in a combination therapy that simultaneouslyattack the tumor along multiple of biochemical pathways. Many of theseregimens, such as the combination of doxorubicin, bleomycin, viblastineand DTIC for Hodgkin's lymphoma, have been developed through empiricaltesting. Because of the inherent limitations of their individualpharmacologic components, such approaches remain relatively non-specificwith high morbidity, allowing considerable room for improvement in termsof efficacy and safety.

Targeting cancers based on their selective overexpression of certaincell-surface receptors or reliance on specific signaling or metabolicpathways, in particular aberrant pathways present in certain cancers,provides another point of attack. For instance, it has been found thatsome cancers harbor mutations in certain protein kinases, such as thoseencoded by the serine/threonine-protein kinase B-Raf gene (BRAF), thatare involved in cell signaling and hyperproliferative growth, therebyserving an oncogene role. Targeting these pathways through the use ofinhibitors has proven attractive, at least initially, in controllingcancers by staving off the oncolytic signaling. A similar approach basedon targeting overexpression of certain receptors, such as epidermalgrowth factor receptor (EGFR) or vascular endothelial growth factor(VEGF), provides the basis for damping the oncolytic activity of thesereceptors, for instance by use of antibodies to the targeted receptors(or by use of agents that inhibit the signaling stimulated by thesereceptors). Unfortunately, as in the case of conventional chemotherapy,these receptors and pathways may play important physiologic rolesperipheral to the tumor, leading to toxicity upon their targeting, whilethe targeted cells also may develop resistance by harnessing alternatebiochemical processes or proliferating via selection of resistant clonalsubpopulations of tumor cells. Thus, the challenges posed by these typesof targeted therapies are substantially similar to those posed byconventional chemotherapy.

In a growing number of oncology indications it is now clear thatcancerous tumors employ various methods to evade detection as aberranttissue and to reduce immune system competency, thereby avoidingpotential identification and destruction by the patient's immune system.As a consequence, a number of approaches have been developed to enhancethe capability of the patient's immune system to detect and destroycancers. For example, the anti-CTLA-4 (cytotoxic T lymphocyte-associatedantigen 4) antibodies ipilimumab and tremelimumab are designed tocounter downregulation of the immune system by blocking CTLA-4 activityand thus augmenting T-cell response against cancer. Alternate approachesmay utilize agents that stimulate certain components of the immunesystem (i.e., upregulation), including administering non-specificcytokines (such as interleukin 1, 2, or 6, “IL-1”, “IL-2” or “IL-6”;interferon-alpha or gamma, “IFN-α” and “IFN-γ”; and granulocytemacrophage colony stimulating factor, “GM-CSF”), or that attempt toprovoke a tumor-specific immune response to certain tumor antigens, suchas dendritic cell vaccines and antibodies against specific tumorantigens and even adoptive T-cell therapy. Additional approaches haveattempted to elicit systemic response following repeated inoculation oftumors with certain immunostimulatory agents, such as an intralesionalvaccine containing an oncolytic herpes virus encoding GM-CSF or aplasmid encoding human leukocyte antigen-B7 and beta-2 microglobulinagent designed to express allogeneic major histocompatibility complex(MHC) class I antigens. For various reasons including, but not limitedto, potential systemic toxicity of these immunomodulating agents,differential expression of the targeted moieties or responsiveness ofclonal subpopulations, increase of tumor burden during therapyinduction, and development of resistance against the selected mode ofattack, current regimens may not result in as robust an immune responseas desired, again allowing considerable room for improvement in terms ofefficacy and safety.

Combination of systemic immunomodulatory agents with systemicchemotherapy agents or kinase inhibitors has been proposed, for exampleby Jure-Kunkel and Lee (WO 2010/014784), however there is limited datato determine whether such an approach will be clinically significant. Inprincipal, this approach combines the features of targeted therapy(using chemotherapy or a metabolic inhibitor) with immunomodulation in acombination therapy, and as is the case with standard chemotherapeuticcombination therapy it provides a means to attack cancer simultaneouslyvia several different paths, thereby increasing potency while reducinglikelihood of resistance. Since the immunologic consequences ofchemotherapy may at least partially counteract the activity of theimmunomodulator, and their respective systemic adverse effects may beadditive or synergistic, such a combination of modalities hassignificant potential shortcomings. While not the topic of Jure-Kunkeland Lee, targeted therapies when combined with immunomodulatory agentscan also have these negative effects. Perhaps most importantly, thesepotential combinations don't appear to afford additive or synergistictumoricidal potency in terms of immunologic benefit since neitherchemotherapy nor metabolic or aberrant gene targeting can be expected tosignificantly activate an antitumor immune response, while the proposedanti-CTLA-4 targeting is similarly unlikely to increase sensitivity oftumor cells to the companion chemotherapy or tumor specific approach.The possibility of increased tumor burden during induction ofimmunomodulatory therapy further complicates the picture, raising thepossibility that the disease may progress to an unacceptably advancedstate during the early phases of the regimen.

Further complicating the therapeutic challenge, tumors that shrinkgradually over a long period of time and slowly release immunoreactivetumor materials in response to any of these conventional systemictherapies may fail to trigger a potent protective response and caninstead facilitate reduced antitumor immunity. This phenomenon issimilar to that underlying low dose therapies for allergies whereby thehost is repeatedly exposed to low doses of antigenic material over aprolonged period, eliciting tolerance by causing the immune system toidentify these persistent “background” antigens as “self” (i.e., anormal part of the host). In a similar fashion, the slow, low doserelease of tumor antigens to the immune system in response to manysystemic therapies may deceive the immune system into tolerance towardtumor antigens thereby reducing or negating possible antitumor response,potentially prolonging tumor survival, and allowing continued metastaticspread.

An alternate class of therapies is predicated on physical restriction ofdelivery of the therapeutic modality to diseased tissue. These localizedtherapies attempt to maximize potency of the therapy within tumor tissuewhile reducing systemic exposure. Approaches include physical orchemical disruption of tumors using intralesional methods, such aspercutaneous ethanol injection therapy (PEIT) and radiofrequency (RF)ablation, and locoregional delivery of potent cytotoxic agents, such asisolated limb perfusion (ILP), isolated limb infusion (ILI) orpercutaneous hepatic perfusion (PHP), with melphalan (an alkylatingagent) or similar agents. While these approaches are often quiteeffective in maximizing pharmacologic activity against the treatedtumor, they have generally exhibited many of the same limitations ofsystemic therapies due to the inherent shortcomings of the underlyingtherapeutic modality, including limited specificity for the targetedcancer with significant locoregional toxicity, and minimal impact onsystemic disease, particularly for those approaches having no mechanismfor immune stimulation against the treated tumor.

The use of cancer-specific cytotoxic agents delivered via anintralesional (IL) route (i.e., IL chemoablation) is a novel hybridapproach that has been described by one or more of the present inventors(for example in U.S. Pat. No. 7,648,695, U.S. Ser. No. 11/951,800 andU.S. Ser. No. 12/315,781, which are incorporated herein in theirentirety). This approach maximizes local efficacy against injectedtumors while minimizing systemic exposure of the patient to the injectedagent and resultant potential for systemic adverse effects. One or moreof the present inventors have shown that IL use of a certain specificclass of agent (for example certain formulations of certain halogenatedxanthenes, exemplified by a 10% (w/v) solution of rose bengal disodiumin saline, termed “PV-10” and undergoing clinical testing for treatmentof metastatic melanoma, breast carcinoma and hepatocellular carcinoma)can elicit not only highly specific ablation of the injected lesion butalso an antitumor immune response (“bystander effect”) that can augmentlocal efficacy in the injected tumor and lead to spontaneous regressionof uninjected tumors. Nonclinical evidence indicates that high levels ofgranulocytes (such as basophils, eosinophils and mast cells) may beexpressed in the tissue surrounding tumors, indicating that the host isattempting to mount a non-specific immune response to tumor tissue.Treatment of tumors with PV-10 can lead to modulation of this responseto one that is more specific and effective (for example, by recruitingmononuclear tumor-infiltrating lymphocytes, TILs, or macrophages intoand around the tumor). It is likely that acute tumor disruptionresulting from IL chemoablation with PV-10 releases sequestered, intacttumor antigens to local antigen-presenting cells (APCs), facilitatingmodulation of the immune response and presentation of appropriateantigenic targets to T and B-cells. Collateral destruction ofgranulocytes surrounding the tumor may precipitate chemokine release andlocal inflammation, and may serve an adjuvant role in promoting specificantitumor response. In situ destruction of the injected tumor assurespresentation of tumor antigens in their natural context, therebymaximizing potential response of the immune system to the treated tumorand to tumors bearing the same immunologic signature. Since immuneresponse is proportional to the intensity and duration of the insult tothe host, the acute exposure achieved through IL chemoablation isimmunologically advantageous relative to the lesser intensity insultproduced by a systemic therapy that is spread out over a long duration,and this acute exposure potentially vaccinates the patient against thetreated tumor.

Acute ablation of the injected tumor also quickly reduces tumor burden,which may be augmented by injecting all or a substantial fraction of apatient's tumors, either in a single treatment session or a series oftreatments fractionated over a period of days or weeks. This may reducethe level of immune suppression exerted by the patient's tumor mass,leading to improved ability of their immune system to mount a successfulattack against remaining tumor tissue. The inherent suitability of ILchemoablation for use against large or multiple cancerous lesions, whenpresent, may further enhance outcome by facilitating in situ inoculationagainst potentially distinct clonal subpopulations in different tumors(or even within individual tumors) that may arise during tumor growthand metastasis.

While IL chemoablation overcomes many of the shortcomings of priortherapeutic modalities (for example by achieving rapid reduction intumor burden, maximizing acute exposure to intact tumor antigens in anappropriate context, and affording minimal potential for systemicadverse effects) one or more of the present inventors have found that itmay not be ideal for all cancer cases, particularly certain advancedcases having rapidly proliferating tumors, those with widelydisseminated disease and those that present in forms that are difficultto fully infiltrate with the IL agent. Accordingly, additionaladvancements are needed in the fields of oncology and improvedtherapeutic regimens therefore.

SUMMARY OF THE INVENTION

The present invention is directed to a method for the treatment ofcancer, said method comprising administration of a therapeuticallyeffective amount of an intralesional chemoablative pharmaceuticalcomposition, or variant of said composition, in combination with atherapeutically effective amount of a systemic immunomodulatoryanticancer agent.

The present invention is also directed to a pharmaceutical compositionfor the treatment of cancer comprising: a therapeutically effectiveamount of an intralesional chemoablative pharmaceutical composition; anda therapeutically effective amount of a systemic immunomodulatoryanticancer agent.

In an embodiment of the above method and pharmaceutical composition, thesystemic immunomodulatory anticancer agent comprises anti-CTLA-4antibodies including ipilimumab and tremelimumab.

In an embodiment of the above method and pharmaceutical composition, thesystemic immunomodulatory anticancer agent is selected from the groupconsisting of non-specific cytokines, such as interleukin-1,interleukin-2, or interleukin-6 (IL-1, IL-2 or IL-6) and aldesleukin;interferon-alpha or interferon-gamma (IFN-α and IFN-γ), interferonalfa-2b and pegylated interferon (including pegylated interferon alfa-2aand pegylated interferon alfa-2b); granulocyte macrophage colonystimulating factor (GM-CSF, molgramostim or sargramostim); dendriticcell vaccines and other allogeneic or autologous therapeutic cancervaccines, including intralesional vaccines containing an oncolyticherpes virus encoding GM-CSF (OncoVex®) or a plasmid encoding humanleukocyte antigen-B7 and beta-2 microglobulin agent designed to expressallogeneic MHC class I antigens (Allovectin-7®); and antibodies againstspecific tumor antigens.

The present invention is also directed to a method for the treatment ofcancer, said method comprising administration of a therapeuticallyeffective amount of an intralesional chemoablative pharmaceuticalcomposition, or variant of said composition, in combination with atherapeutically effective amount of a systemic targeted anticanceragent.

The present invention is also directed to a pharmaceutical compositionfor the treatment of cancer comprising: a therapeutically effectiveamount of an intralesional chemoablative pharmaceutical composition; anda therapeutically effective amount of a systemic targeted anticanceragent.

In an embodiment of the above method and pharmaceutical composition, thesystemic targeted anticancer agent is selected from the group consistingof drugs that target protein kinases and the receptors that activatethem, including afatinib (BIBW 2992), bevacizumab, cetuximab, dasatinib,E7080, erlotinib, gefitinib, imatinib, lapatinib, nilotinib,panitumumab, pazopanib, pegaptanib, ranibizumab, sorafenib, sunitinib,trastuzumab and vandetanib; serine/threonine-selective protein kinaseinhibitors, including those targeting the B-Raf/MEK/ERK pathway, such asvemurafenib (also known as PLX4032, RG7204 or RO5185426), GSK2118436 andGSK1120212; aromatase inhibitors, including aminoglutethimide,anastrozole, exemestane, fadrozole, formestane, letrozole, testolactoneand vorozole; estrogen receptor antagonists, including lasofoxifene,raloxifene, tamoxifen and toremifene; COX-2 inhibitors, includingcelecoxib, valdecoxib and rofecoxib; angiogenesis blockers, includingIFN-α, IL-12, suramin, and thrombospondin (including thrombospondin 1,ABT-510 and ABT-898); and immune cell therapy, including adoptive T-celltransfer and autologous immune cell therapy.

In a further embodiment of all of the above methods and pharmaceuticalcompositions, the intralesional chemoablative pharmaceutical compositioncomprises an IL chemoablative agent comprising primarily a halogenatedxanthene in an appropriate pharmaceutical composition, including a 0.1%(w/v) or higher concentration aqueous solution of the halogenatedxanthene or mixtures thereof, or a physiologically acceptable salt ofthe halogenated xanthene.

In a further embodiment of all of the above methods and pharmaceuticalcompositions, the halogenated xanthene is rose bengal(4,5,6,7-tetrachloro-2′,4′,5′,7′-tetraiodofluorescein).

In a further embodiment of all of the above methods and pharmaceuticalcompositions, the halogenated xanthene is rose bengal disodium.

In a further embodiment of all of the above methods and pharmaceuticalcompositions, the halogenated xanthene is selected from the groupconsisting of erythrosin B, phloxine B,4,5,6,7-tetrabromo-2′,4′,5′,7′-tetraiodofluorescein,2′,4,5,6,7-pentachloro-4′,5′,7′-triiodofluorescein,4,4′,5,6,7-pentachloro-2′,5′,7′-triiodofluorescein,2′,4,5,6,7,7′-hexachloro-4′,5′-diiodofluorescein,4,4′,5,5′,6,7-hexachloro-2′,7′-diiodofluorescein,2′,4,5,5′,6,7-hexachloro-4′,7′-diiodofluorescein,4,5,6,7-tetrachloro-2′,4′,5′-triiodofluorescein,4,5,6,7-tetrachloro-2′,4′,7′-triiodofluorescein,4,5,6,7-tetrabromo-2′,4′,5′-triiodofluorescein, and4,5,6,7-tetrabromo-2′,4′,7′-triiodofluorescein.

In a further embodiment of all of the above methods and pharmaceuticalcompositions, the halogenated xanthene has a concentration of about 0.1%(w/v) up to about 20% (w/v), and that the pharmaceutical compositionincludes an electrolyte comprising at least one cation selected from thegroup consisting of sodium, potassium, calcium and magnesium and atleast one anion selected from the group consisting of chloride,phosphate and nitrate, wherein the electrolyte is at a concentration ofbetween about 0.1% (w/v) and about 2% (w/v).

In a further embodiment of all of the above methods and pharmaceuticalcompositions, the concentration of said electrolyte in the ILchemoablative pharmaceutical composition is between 0.5 to 1.5% (w/v).

In a further embodiment of all of the above methods and pharmaceuticalcompositions, the chemoablative pharmaceutical composition has anosmolality of the composition of greater than about 100 mOsm/kg.

In a further embodiment of all of the above methods and pharmaceuticalcompositions, the electrolyte is sodium chloride.

In a further embodiment of all of the above methods and pharmaceuticalcompositions, the pharmaceutical composition comprises a hydrophilicvehicle.

In a further embodiment of all of the above methods and pharmaceuticalcompositions, the pharmaceutical composition has a pH in the range ofbetween about 4 to about 10.

In a further embodiment of all of the above methods and pharmaceuticalcompositions, the pharmaceutical composition has a pH in the range ofbetween about 5 to about 7.

In a further embodiment of all of the above methods and pharmaceuticalcompositions, the methods and pharmaceutical compositions are for thetreatment of cancers selected from melanoma, breast cancer, primary andmetastatic liver cancer, prostate cancer and small cell and non smallcell lung cancer.

DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS

One aspect of the present invention is the result of unanticipatedsynergy resulting upon combination of certain local therapeuticmodalities, and in particular certain local immunomodulative therapiessuch as for example IL chemoablation with PV-10 or another halogenatedxanthene agent, with certain systemic therapeutic modalities. Thiscombination can boost the therapeutic activity of both therapeuticmodalities with the potential for no significant increase, or even anoverall decrease, in morbidity relative to that typically achieved usingthe component therapies separately.

One or more of the present inventors have shown that IL chemoablationcan lead to rapid reduction in a patient's tumor burden, reducingpotential for tumor-induced immune suppression, extent and severity ofthe disease, and continued drag on the patient's immune and otherphysiologic functions. The resultant acute exposure of the patient'simmune system to intact tumor antigens in proper biological context ismarkedly different from that achieved using systemic chemotherapy,targeted systemic therapies, or other local therapeutic modalities, eachof which generally produce at best a gradual, low level chronic exposureof the immune system to tumor antigens, often in an inappropriatecontext. Chemoablation of entire tumors or substantially the entirety oftumors, and especially chemoablation of multiple tumors, enhancesexposure of the patient's immune system to any distinct clonalsubpopulations of tumor cells that may be present, maximizing overallresponse to the in situ antitumor vaccination. Hence, theimmunomodulatory effects achieved may be superior in breadth and potencyto those achieved using prior therapeutic approaches.

However, for cases where disease is rapidly proliferating, or is widelydisseminated, or presents in a form difficult to fully infiltrate withthe IL chemoablative agent, use of complementary therapeutic modalitiesoffers additive or synergistic benefit, particularly when theycontribute immunologic stimulation (i.e., immunodulation) thatcomplements that afforded through IL chemoablation. The use of suchcomplementary immunomodulative therapies may have further advantage interms of additive or synergistic immunologic interactions that allow oneor both therapies to be used at reduced doses (relative to that neededwhen used individually as monotherapies) while retaining high efficacy,thereby reducing undesirable adverse effects.

In particular, the use of a potent local immunomodulative therapy, suchas IL chemoablation with, for example, PV-10 or another halogenatedxanthene agent, in conjunction with one or more systemicimmunomodulative therapies (especially those that elicit immune systemupregulation or counter tumor-induced immune system down regulation) ishighly attractive since this combination yields a uniquely salubriouscombination: exposure of the patient's potentiated immune system to theintense antigenic “insult” produced upon IL chemoablation. The effectsof such combination may be heightened by potentiation of the immunesystem at the time of chemoablation or subsequent to chemoablation.Since IL chemoablation is well suited to repeat treatment, continuedpotentiation of the patient's immune system, for example by continuedadministration of the systemic immunomodulatory therapy, while ILchemoablation is administered one or more times, is a preferredembodiment. As an alternate embodiment, IL chemoablation may be followedby commencement of systemic immunomodulatory therapy, for example aftera delay of several weeks or more when a reduction in local inflammationor other non-specific immunologic effects is desirable.

The potential of benefits of combining local immunomodulatory therapywith a systemic immunomodulatory therapy regimen may make otherwiseundesirable systemic immunomodulatory therapies viable: due to theresultant augmentation in potency of the systemic component of thecombination therapy, reduced systemic dose regimens may be possible withcommensurate reduction in adverse effects from the systemic therapy.Further, since the adverse effect profile of the local immunomodulatorytherapy (i.e., IL chemoablation) is orthogonal to that of most systemicimmunomodulatory therapies, a combined local and systemicimmunomodulatory therapy is inherently safer and more attractivecompared with prior combinations that can produce undesirable additiveor synergistic adverse effects.

The combination of massive exposure to tumor antigens coupled withreduced tumor burden that results from IL chemoablation is particularlyattractive in this context, since it maximizes potential immuneactivation while diminishing potential immune downregulation andphysiologic demand from the tumor mass. When combined with a systemictherapy that further enhances immunologic upregulation or reducesdownregulation, the effects on antitumor immunity, both at the ablatedlesion and at uninjected sites, including those proximal and distant tothe injection sites, will be additive or synergistic.

Many of the advantages accrued upon combining local immunomodulatorytherapy with a systemic immunomodulatory therapy may be achieved throughsimilar combination of local immunomodulatory therapy with a systemictargeted therapy, such as IL chemoablation combined with a targetedkinase inhibitor. Since IL chemoablation has a uniquely disruptiveeffect on tumor tissue, combination of this modality with an approachthat targets tumor viability via an orthogonal path, such as those thattarget aberrant growth signaling or overexpression of receptors involvedin tumor hyperproliferation, can yield enhanced efficacy in the treatedtumor. For example, by using a systemic targeted therapy to increasestress on the tumor or reduce tumor viability in the wake of ILchemoablation, the cytotoxicity of the IL treatment may be enhanced atthe time of IL treatment; response of any remaining tumor tissue mayalso be increased to immunologic activation resulting from the ILtreatment since the systemic therapy will counter proliferation ofresidual tumor tissue without interfering with development of the immuneresponse from chemoablation. The rapid reduction in tumor burdenresulting from IL chemoablation further augments these advantages byreducing immune suppression and physiologic demands from the tumortissue. Since the systemic targeted therapy is not required to achievecomplete control or eradication of substantial tumor masses in thiscontext, but rather serves to augment the activity of the localimmunomodulatory therapy, it may be possible to administer the systemictherapy at a reduced dose, thereby minimizing potential adverse effectsand making the combined therapy safer and more attractive compared withprior systemic combinations. Addition of the immunologic responseresulting from the local immunomodulatory therapy component provides ameans to counter resistance problems that have plagued many targetedsystemic therapies, such as the BRAF inhibitors, particularly whencontinuous systemic therapies are required to maintain long term controlof the disease, since long term control will result from the immuneresponse rather than perpetual reliance on the targeted systemictherapy.

In some cases it may be desirable to commence systemic targeted therapyprior to local immunomodulatory therapy, for instance when diseaseburden is very high or widespread, or when the disease is rapidlyproliferating, potentially making effective administration of the localimmunomodulatory therapy difficult or less effective. In this manner,the systemic targeted therapy may be used to control or reduce tumorburden prior to administration of local immunomodulatory therapy inorder to enhance responsiveness of the disease to the localimmunomodulatory therapy. Such an approach is tantamount to “downstaging” disease status prior to commencement of local immunomodulatorytherapy. For example, certain BRAF inhibiting drugs have proveneffective at temporarily reducing disease burden in advanced stagemetastatic melanoma, but resistance often develops within a period ofmonths, negating long term outcome. Treatment of residual disease withlocal immunomodulatory therapy, such as IL chemotherapy, while itremains under control of the targeted therapy provides a means forelimination of residual tumor burden while stimulating long termimmunity to recurrence, thereby improving ultimate outcome.

Problems affecting attempts to develop and utilize therapeutic cancervaccines may also be similarly mitigated or resolved through combinationof such vaccines with local immunomodulatory therapy. Specifically, lackof survival benefit observed in clinical trials of some such vaccines inadvanced stage cancer, such as Canvaxin for stage III or IV melanoma,appears to be due in part to failure of vaccination to overcome existingtumor burden present in patients at the time of vaccination andcontinued increase in their disease level during the induction intervalnecessary for development of an immune response from vaccination. As inthe case with systemic targeted therapy, the rapid reduction in tumorburden resulting from IL chemoablation can mitigate suppression of theimmune system by the patient's disease burden while providing criticaltime for onset of the immune response from vaccination, therebymaximizing potential local and systemic antitumor effects through thecombined action of the local immunomodulatory therapy and systemicvaccination.

Examples of combination therapies and method of treatment within thepresent invention include but are not limited to the following:

Local immunomodulative therapy combined with one or more systemicinhibitor of immune system down regulation, such as anti-CTLA-4antibodies including but not limited to ipilimumab and tremelimumab.

Local immunomodulative therapy combined with one or more systemic immuneupregulating agent, including: non-specific cytokines, such asinterleukin-1, -2, or -6 (IL-1, IL-2 or IL-6) and aldesleukin;interferon-alpha or gamma (IFN-α and IFN-γ), interferon alfa-2b andpegylated interferon (including pegylated interferon alfa-2a andpegylated interferon alfa-2b); granulocyte macrophage colony stimulatingfactor (GM-CSF, molgramostim or sargramostim); dendritic cell vaccinesand other allogeneic or autologous therapeutic cancer vaccines,including intralesional vaccines containing an oncolytic herpes virusencoding GM-CSF (OncoVex®)or a plasmid encoding human leukocyteantigen-B7 and beta-2 microglobulin agent designed to express allogeneicMHC class I antigens (Allovectin-7®); and antibodies against specifictumor antigens.

Local immunomodulative therapy combined with one or more systemictargeted therapy agent, including: drugs that target protein kinases andthe receptors that activate them, including but not limited to afatinib(BIBW 2992), bevacizumab, cetuximab, dasatinib, E7080, erlotinib,gefitinib, imatinib, lapatinib, nilotinib, panitumumab, pazopanib,pegaptanib, ranibizumab, sorafenib, sunitinib, trastuzumab andvandetanib; serine/threonine-selective protein kinase inhibitors,including but not limited to those targeting the B-Raf/MEK/ERK pathway,such as vemurafenib (also known as PLX4032, RG7204 or RO5185426),GSK2118436 and GSK1120212; aromatase inhibitors, including but notlimited to aminoglutethimide, anastrozole, exemestane, fadrozole,formestane, letrozole, testolactone and vorozole; estrogen receptorantagonists, including but not limited to lasofoxifene, raloxifene,tamoxifen and toremifene; COX-2 inhibitors, including but not limited tocelecoxib, valdecoxib and rofecoxib; angiogenesis blockers, includingIFN-α, IL-12, suramin, and thrombospondin (including thrombospondin 1,ABT-510 and ABT-898); and immune cell therapy, including but not limitedto adoptive T-cell transfer and autologous immune cell therapy.

Typically, monotherapy dose schedules are set by determining the maximumtolerated dose (MTD) in early-stage clinical trials. The MTD (or a closevariation thereon) is then promulgated to later-stage clinical trialsfor assessment efficacy and more detailed assessment of safety. TheseMTDs frequently become the established therapeutic dose upon completionof clinical testing. Example dosing schedules for a number of systemicagents that may be combined in the present invention with localimmunomodulative therapy are provided in Table 1.

TABLE 1 Example systemic immunomodulatory or targeted anticancer agentsSystemic Agent Typical Dose Schedule Ipilimumab 3 mg/kg q21d for 4treatments Tremelimumab 15 mg/kg q3m Aldesleukin 600,000 IU/kg q8h (upto 14 doses before 9 day rest and repeat; rest at least 7 wks beforerepeat of course) interferon alfa-2b 20 million IU/m² 5 times per weekfor 4 weeks (induction phase) followed by 10 million IU/m² three timesper week (maintenance phase) pegylated interferon 6 μg/kg qwk for eightweeks (induction phase) followed by 3 μg/kg qwk (maintenance phase)Oncovex ® 4 mL IL at 10⁸ pfu/mL GM-CSF 125 μg/m² daily for 14 wksfollowed by 14 days rest Allovectin-7 ® 2 mg IL qwk for 6 wks Afatinib20-50 mg daily Bevacizumab 5-15 mg/kg q14d-q21d Cetuximab 400 mg/m²followed by weekly maintenance at 250 mg/m² Dasatinib 100 mg dailyErlotinib 100-150 mg daily Gefitinib 250 mg daily Imatinib 400-600 mgdaily (increased to twice daily if well tolerated or disease progresses)Lapatinib 1250 mg daily for 21 day cycle Nilotinib 400 mg twice dailyPanitumumab 6 mg/kg q14d Pazopanib 800 mg daily Pegaptanib 0.3 mg q6wksRanibizumab 0.5 mg q4wks Sorafenib 400 mg twice daily Sunitinib 50 mgdaily for 4 weeks followed by 2 week recovery Trastuzumab 4 mg/kgfollowed by weekly maintenance at 2 mg/kg Vandetanib 200-300 mg dailyVemurafenib 960 mg twice daily (PLX4032) GSK2118436^(a) 150 mg twicedaily GSK1120212^(a) 2 mg daily aminoglutethimide 250 mg q6h Anastrozole1 mg daily Exemestane 25 mg daily Fadrozole 1 mg twice daily Formestane250 mg daily Letrozole 2.5 mg daily Vorozole 2.5 mg daily Raloxifene 60mg daily Tamoxifen 20-40 mg daily Toremifene 60 mg daily Celecoxib200-400 mg twice daily Rofecoxib 20-25 mg daily Suramin 1 g qwkthrombospondin 20 mg daily to 100 mg twice daily (ABT-510^(a))^(a)Proprietary code name for drug under development for which nononproprietary name is currently available.

Because of additive or synergistic effects, the combination therapiesand method of treatment of the present invention will generally allowuse of the systemic agent at a level at or below the typical doseschedule for the systemic agent, such as those described in Table 1,when used with a local immunomodulative therapy, such as that describedinfra.

Local immunomodulative therapy includes but is not limited tointralesional chemoablation using an IL chemoablative agent consistingprimarily of rose bengal(4,5,6,7-tetrachloro-2′,4′,5′,7′-tetraiodofluorescein) or anotherhalogenated xanthene, including erythrosin B, phloxine B,4,5,6,7-tetrabromo-2′,4′,5′,7′-tetraiodofluorescein,2′,4,5,6,7-pentachloro-4′,5′,7′-triiodofluorescein,4,4′,5,6,7-pentachloro-2′,5′,7′-triiodofluorescein,2′,4,5,6,7,7′-hexachloro-4′,5′-diiodofluorescein,4,4′,5,5′,6,7-hexachloro-2′,7′-diiodofluorescein,2′,4,5,5′,6,7-hexachloro-4′,7′-diiodofluorescein,4,5,6,7-tetrachloro-2′,4′,5′-triiodofluorescein,4,5,6,7-tetrachloro-2′,4′,7′-triiodofluorescein,4,5,6,7-tetrabromo-2′,4′,5′-triiodofluorescein, and4,5,6,7-tetrabromo-2′,4′,7′-triiodofluorescein in an appropriatepharmaceutical composition, including a 0.1% (w/v) or higherconcentration aqueous solution of rose bengal (i.e., PV-10) orequivalent solution of another halogenated xanthene or mixtures thereof.A physiologically acceptable salt of the halogenated xanthene may beused in this composition.

The present invention includes immunotherapeutic procedures whereinlarge amounts of tumor antigen are exposed to a patient's immune system,for example upon intralesional delivery of an immunomodulator, includingbut not limited to intralesional rose bengal, in combination with one ormore systemic immunomodulator, to enhance the immune-mediated antitumorresponse.

About the preferred IL chemoablative agents:

Local immunomodulative therapy includes, as a preferred embodiment,intralesional chemoablation using rose bengal or another halogenatedxanthene. A preferred form, rose bengal disodium, has the followingformula:

Certain details of this preferred embodiment for the localimmunomodulative composition are described in Applicants' co-pendingapplication U.S. Ser. No.12/315,781, which is incorporated herein in itsentirety. This preferred embodiment of the present invention isdescribed here with particular relevance to melanoma. However, thepresent invention may also find application for the treatment of otherhyperproliferative diseases including, but not limited to, cancers, suchas for example, breast cancer, primary and metastatic liver cancer,prostate cancer and small cell and non small cell lung cancer, and nolimitation is intended thereby.

Malignant melanoma is the most serious form of skin cancer and accountsfor 80% of skin cancer deaths.

The extent of spread of a disease is described by stages. Stage 0melanoma is a very early stage disease known as melanoma in situ.Patients with melanoma in situ are classified as Tis (tumor in situ).The tumor is limited to the epidermis with no invasion of surroundingtissues, lymph nodes, or distant sites. Melanoma in situ is consideredto be very low risk for disease recurrence or spread to lymph nodes ordistant sites. Treatment is by surgical excision with a margin ofhealthy skin.

In stage I melanoma, the tumor has penetrated in to the skin by lessthan 1mm but has not spread. Treatment is by wide local excision and theprobability of disease free survival in five years is between 90 to 95%.

Stage II melanoma describes a tumor that has penetrated more than 1 mminto the skin but has not spread. Wide local excision is the preferredtreatment. However, excision at this stage carries a much higher riskand less favorable prognosis than excision of a Stage I tumor.

Stage III melanoma is characterized by the existence of one or morenodal, in-transit or satellite metastasis but has not spread to distantor visceral sites. In-transit metastases are distant from the primarytumor but not reaching the draining nodal basin. Satellite metastasesare intralymphatic extensions of the primary tumor and are typicallyfound closer to the primary tumor than in-transit metastasis. Five yearsurvival for stage III patients ranges from approximately 24% (grossnodal disease) to 80% (microscopic nodal disease).

Stage IV melanoma is when the disease has spread to distant sites.Survival of stage IV melanoma drops to approximately 10%.

Similar staging systems exist for all major cancers, and are generallybased on the clinical presentation and histopathologic details of thedisease and how these factors have been shown to impact survival.

Standard treatment for easily removable Stage III tumors is wide areaexcision together with removal of lymph nodes. Adjunct treatment such asradiotherapy and chemotherapy and for regional limb metastases, regionalinfusion of melphalan or other chemotherapeutic agents may also begiven. However, in some cases, surgery is contraindicated due to thenumber and/or location of tumors and other treatment options must beconsidered. Unfortunately, response levels for these other options arenot high. For example, melanoma is largely resistant to radiationtherapy. Systemic chemotherapy also has modest response rates againstmelanoma. The most effective chemotherapy regimen to-date issingle-agent dacarbazine, which is only successful in 10-15% of cases.Two combination chemotherapy regimens commonly used in the treatment ofpatients with advanced-stage melanoma are the cisplatin, vinblastine andDTIC (CVD) regimen and the Dartmouth regimen, which is a combination ofcisplatin, DTIC, carmustine and tamoxifen.

When melanoma occurs in the extremities, chemotherapy agents may bedelivered via hyperthermic isolated limb perfusion (ILP). With thistechnique, blood vessels are accessed surgically, the blood flow to andfrom the limb is stopped using a tourniquet, and a warmed solution ofchemotherapy drug is administered directly into the blood of the limb,allowing higher doses of drugs to be dispensed than with systemictreatment. A less invasive regional therapy is isolated limb infusion(ILI) whereby vascular access is gained via a percutaneous route in thegroin.

Another treatment option is intralesional therapy in which achemotherapeutic agent is injected directly into the tumor. BacilleCalmette Guerin (BCG) was one of the earliest reagents used for ILtherapy. A review of data from 15 trials found 19% complete response(CR) and 26% partial response (PR) with extended survival in 13% ofstage III patients.

IL interferons (IFN) have yielded mixed results ranging from a report of45% objective response rate (ORR, 31% CR+14% PR) for IFN-α to either noresult or transient response with IFN-γ. Both regimes producedsignificant toxicity and side effects.

IL interleukin-2 appears to be the most promising IL therapy to datewith an ORR in 83% of patients (62% CR+21% PR) receiving 2-3 weekly ILtreatments. Some patients reported flu like symptoms and some authorsnoted that although new lesions appeared during the course of treatment,some patients experienced a marked slowing of the appearance of newcutaneous lesions.

IL therapy with cisplatin or IL cisplatin with electroporation hasyielded results ranging from 38% ORR (19% CR+19% PR) to 53% ORR (47%CR+7% PR). However, the ORR reported for lesions with a median diameterof 0.6 cm of 53% decreased to 44% for lesions having a median diameterof 3.0 cm.

Substantial efficacy has been reported upon a single electrochemotherapytreatment with IL bleomycin. However, as with cisplatin, response wasgenerally reduced in larger tumors.

It may be appreciated that there remains a need for alternative methodsfor the treatment of hyperproliferative diseases and in particular stageIII and IV melanoma.

According to a preferred embodiment of the present invention, there isprovided a method for the treatment of cancer in a patient, such asmetastatic melanoma, the method comprising treatment of the cancerpatient with a local immunomodulative therapy combined with one or moresystemic immunomodulatory therapy or systemic targeted therapy, whereinsaid local immunomodulatory therapy comprises intralesionaladministration of a chemoablative pharmaceutical composition comprisinga hydrophilic vehicle containing4,5,6,7-Tetrachloro-2′,4′,5′,7′-tetraiodofluorescein (i.e. rose bengal),or certain other halogenated xanthene, or a physiologically acceptablesalt thereof. It is preferred that the halogenated xanthene be presentin this pharmaceutical composition at a concentration of about 0.1%(w/v) up to about 20% (w/v), and that the pharmaceutical compositioninclude an electrolyte comprising at least one cation selected from thegroup consisting of sodium, potassium, calcium and magnesium and atleast one anion selected from the group consisting of chloride,phosphate and nitrate, wherein the electrolyte is at a concentration ofbetween about 0.1% (w/v) and about 2% (w/v). It is also preferred thatthe pH of the pharmaceutical composition be between about 4 to about 10.

The term “physiologically acceptable salt” refers to any non-toxicalkali metal, alkaline earth metal, and ammonium salt commonly used inthe pharmaceutical industry, including the sodium, potassium, lithium,calcium, magnesium, barium, ammonium and protamine zinc salts, which canbe prepared by methods known in the art. Preferably, the salts aresodium, potassium, calcium and ammonium in either the mono or dibasicsalt form.

Especially preferred in this IL chemoablative pharmaceutical compositionis the disodium salt of rose bengal. Previous work by one or more of thepresent inventors (WO 02/05812) reported their discovery that rosebengal exhibits preferential uptake into cancer cells but is essentiallyexcluded from normal cells.

One or more of the present inventors have also reported their discoverythat the nature of the vehicle in which the halogenated xanthene, or aphysiologically acceptable salt thereof, is administered cansignificantly influence the degree of partitioning into tumor cells. Inparticular, one or more of the present inventors have surprisinglydiscovered that at an electrolyte concentration of between about 0.1%(w/v) and about 2.0% (w/v), partitioning into tumor cells may rapidly beincreased.

An approximation of an agent's potential for tissue accumulation can beestimated based upon the partition coefficient K_(p). This in vitroparameter is purported to have predictive values relating to in vitrodelivery at the cellular level. In particular, a value greater thanunity is considered to indicate agents capable of localizing in tissue,and thereby being capable of exhibiting enhanced chemotherapeuticefficacy in such tissue. One or more of the present inventors surmisethat values much greater than approximately 50-100 may indicate excesslipophilicity (tendency to accumulate in organic environments) that maycompromise delivery of an agent in a desirable aqueous (i.e.,hydrophilic) formulation. K_(p) is determined by measuring the ratio ofequilibrium concentrations of an agent in a lipophilic phase (n-octanol)contacted with an aqueous phase.

One or more of the present inventors have also reported their discoverythat it is preferred that the pH of the IL chemoablative pharmaceuticalcomposition is in the range of between about 4 to about 10, and morepreferably between about 5 to about 9, to yield maximum solubility ofthe halogenated xanthene in an aqueous vehicle and assure compatibilitywith biological tissue. A particularly preferred pH is between about 4to about 7, preferably between about 5 to about 7, more preferablybetween about 6 to about 7. At these pH values, the halogenatedxanthenes generally remain in dibasic form, rather than the waterinsoluble lactone that forms at low pH.

The pH of the IL chemoablative pharmaceutical composition may beregulated or adjusted by any suitable means known to those of skill inthe art. The composition may be buffered or the pH adjusted by additionof acid or base or the like. As the halogenated xanthenes, orphysiologically acceptable salts thereof, are weak acids, depending uponhalogenated xanthene concentration and/or electrolyte concentration, thepH of the composition may not require the use of a buffer and/or pHmodifying agent. It is especially preferred, however, that thecomposition does not contain any buffer, allowing it to conform to thebiological environment once administered.

One or more of the present inventors have also reported their discoverythat K_(p) is also dependent upon electrolyte concentration with theK_(p) value increasing with electrolyte concentration. Particularlypreferred concentrations of electrolyte in the IL chemoablativepharmaceutical composition are between 0.5 to 1.5% (w/v), and even morepreferably at a concentration of about 0.8 to 1.2% (w/v) and mostpreferably at a concentration of about 0.9% (w/v), this latterconcentration being especially preferred since it corresponds to anapproximately isotonic solution.

In a further preferred embodiment of the present invention, theelectrolyte in the IL chemoablative pharmaceutical composition is sodiumchloride.

Electrolytes at such levels increase the osmolality of the ILchemoablative pharmaceutical composition. Thus, as an alternative tospecifying a range of electrolyte concentrations, osmolality may be usedto characterize, in part, the electrolyte level of the composition. Itis preferred that the osmolality of the composition be greater thanabout 100 mOsm/kg, and more preferably that the osmolality of thecomposition be greater than about 250 mOsm/kg and most preferably thatit is about 300-500 mOsm/kg.

One or more of the present inventors have found that the preferredconcentration of halogenated xanthene and/or dose of IL chemoablativepharmaceutical composition will be dependent upon factors including, butnot limited to, tumor size, number and location. For visceral or otherinternal lesions, such as cancers of the liver, intralesionaladministration may be by percutaneous or intraoperative administration.

One or more of the present inventors have also found that halogenatedxanthene concentrations in the IL chemoablative pharmaceuticalcomposition above about 1% (w/v) to 3% (w/v) are particularly useful forchemoablative use, since lower concentrations are generally insufficientto directly elicit destruction of target tissues. Thus, in a preferredembodiment, the concentration of halogenated xanthene is in the range offrom about 3% (w/v) to about 20% (w/v). In another embodiment, theconcentration of halogenated xanthene is from about 3% (w/v) to about10% (w/v). In another embodiment, the concentration of halogenatedxanthene is from about 10% (w/v) to about 20% (w/v). In still anotherembodiment, the concentration of halogenated xanthene is about 10%(w/v). One or more of the present inventors have surprisingly found thatat these concentrations, not only can an efficient therapeutic responsebe obtained, but the solution is also highly stable and can be readilyhandled both in manufacture and use. These preferred concentrations maybe expressed in weight to volume (w/v), however, concentration in weightto weight (w/w) is substantially equivalent.

Typical dosages of the IL chemoablative pharmaceutical compositionadministered by IL administration range from between 0.1 mL/cc lesionvolume to about 2 mL/cc lesion volume, most preferably between about0.25 mL/cc to about 0.75 mL/cc lesion volume. Such doses typicallycorrespond to a patient dose of between about 10 mg to about 1500 mg ofhalogenated xanthene (which are significantly higher than those dosesused for diagnostic liver tests).

Since the pharmaceutical composition is for IL administration, which isan intracorporeal route, it is further preferred that it be sterile,such as required for conformance to U.S. Pharmacopeia (USP) test <71>,and further that it contains negligible levels of pyrogenic material,such that it conforms to USP<85> (limulus amebocyte lysate assay) or toUSP <151> (rabbit pyrogen test), or to substantially equivalentrequirements, at a pyrogen or endotoxin level equivalent to not morethat (NMT) 10 endotoxin units (EU) per mL. Moreover, the pharmaceuticalcomposition should conform to requirements limiting content ofparticulate matter as defined in USP <788> (i.e., NMT 3000 particulatesgreater than 10 microns in size, and NMT 300 particulates greater than25 microns in size, per container) or substantially equivalentrequirements. Each of these references from the USP is incorporatedherein by reference.

Still further, one or more of the present inventors have found that ahydrophilic vehicle is preferred for the pharmaceutical composition tomaximize preference of the halogenated xanthene for partitioning intocancerous tissue. Accordingly, it is preferred that the pharmaceuticalcomposition contains a minimum of non-hydrophilic components that mightinterfere with such partitioning. It is preferred that the hydrophilicvehicle is water, and it is most preferred that this pharmaceuticalcomposition consists substantially of water.

One or more of the present inventors have found that such pharmaceuticalcompositions as described herein are optimally packaged in glass vialshaving a capacity of approximately 1 to 10 mL, and more preferablyapproximately 5 mL. Such capacities are well suited as unidose forms(i.e., single use packages) for IL treatments.

In a preferred embodiment, the formulation of the pharmaceuticalcomposition is not buffered. In this case, it is preferred thatpackaging containers be made of the USP Type I (low extractable orchemically resistant borosiciliate) or USP Type II (low-extractable sodalime) glass and that the inside surface of such glass containers besurface treated to reduce surface alkalinity of the container that couldadversely affect pH or long-term stability. Typical surface treatmentapplicable to such containers is described in USP<661>. The inside ofsuch surface-treated glass containers should be rinsed with a suitablesolvent, such as distilled water one or more times prior to filling inorder to remove any residue of such surface treatment. The containersshould also be depyrogenated prior to filling, for example, by heatingto 250° C. or higher for several hours or more, and should be sterile orsterilized prior to filling using methods common in the field. If isfurther preferred that such containers have a minimum neck size, forexample, of less than 10 mm and more preferably 5 mm or less, to reducesurface area of the closures of the containers (and hence exposure ofthe medicament to such closures).

One or more of the present inventors have further found that aseptum-type closure, composed preferably of a pharmaceutical gradeelastomeric material with a Teflon or similar inner coating, isparticularly suitable for use with the IL chemoablative pharmaceuticalcomposition since it facilitates insertion of a needle into thecontainer for withdrawal of a dose of medicament while exhibitingminimal potential for interaction with the container contents.

It is also preferred that the pharmaceutical composition does notinclude any preservatives. One or more of the present inventors havefound that it is generally preferable to avoid use of preservatives,many of which may deleteriously interfere with the pharmaceuticalcomposition or formulation thereof, or may complex or otherwise interactwith or interfere with the delivery of the halogenated xanthene activecomponent. To the extent that a preservative may be used, one or more ofthe present inventors have found that imidurea is preferred as it doesnot interact with halogenated xanthenes, either in the pharmaceuticalcomposition or upon administration.

This description has been offered for illustrative purposes only and isnot intended to limit the invention of this application.

1. A method of treatment of cancer comprising administering atherapeutically effective amount of an intralesional chemoablativepharmaceutical composition in combination with a therapeuticallyeffective amount of a systemic immunomodulatory anticancer agent.
 2. Themethod of claim 1 wherein said systemic immunomodulatory anticanceragent comprises anti-CTLA-4 antibodies including ipilimumab andtremelimumab.
 3. The method of claim 1 wherein said systemicimmunomodulatory anticancer agent is selected from the group consistingof non-specific cytokines, such as interleukin-1, interleukin-2, orinterleukin-6 (IL-1, IL-2 or IL-6) and aldesleukin; interferon-alpha orinterferon-gamma (IFN-α and IFN-γ), interferon alfa-2b and pegylatedinterferon (including pegylated interferon alfa-2a and pegylatedinterferon alfa-2b); granulocyte macrophage colony stimulating factor(GM-CSF, molgramostim or sargramostim); dendritic cell vaccines andother allogeneic or autologous therapeutic cancer vaccines, includingintralesional vaccines containing an oncolytic herpes virus encodingGM-CSF (OncoVex®) or a plasmid encoding human leukocyte antigen-B7 andbeta-2 microglobulin agent designed to express allogeneic MHC class Iantigens (Allovectin-7®); and antibodies against specific tumorantigens.
 4. The method of claim 1 wherein said intralesionalchemoablative pharmaceutical composition comprises an IL chemoablativeagent comprising primarily a halogenated xanthene in an appropriatepharmaceutical composition, including a 0.1% (w/v) or higherconcentration aqueous solution of the halogenated xanthene or mixturesthereof, or a physiologically acceptable salt of the halogenatedxanthene.
 5. The method of claim 4 wherein the halogenated xanthene isrose bengal (4,5,6,7-tetrachloro-2′,4′,5′,7′-tetraiodofluorescein). 6.The method of claim 5 wherein the halogenated xanthene is rose bengaldisodium.
 7. The method of claim 4 wherein the halogenated xanthene isselected from the group consisting of erythrosin B, phloxine B,4,5,6,7-tetrabromo-2′,4′,5′,7′-tetraiodofluorescein,2′,4,5,6,7-pentachloro-4′,5′,7′-triiodofluorescein,4,4′,5,6,7-pentachloro-2′,5′,7′-triiodofluorescein,2′,4,5,6,7,7′-hexachloro-4′,5′-diiodofluorescein,4,4′,5,5′,6,7-hexachloro-2′,7′-diiodofluorescein,2′,4,5,5′,6,7-hexachloro-4′,7′-diiodofluorescein,4,5,6,7-tetrachloro-2′,4′,5′-triiodofluorescein,4,5,6,7-tetrachloro-2′,4′,7′-triiodofluorescein,4,5,6,7-tetrabromo-2′,4′,5′-triiodofluorescein, and4,5,6,7-tetrabromo-2′,4′,7′-triiodofluorescein.
 8. The method of claim 4wherein said halogenated xanthene has a concentration of about 0.1%(w/v) up to about 20% (w/v), and that the pharmaceutical compositionincludes an electrolyte comprising at least one cation selected from thegroup consisting of sodium, potassium, calcium and magnesium and atleast one anion selected from the group consisting of chloride,phosphate and nitrate, wherein the electrolyte is at a concentration ofbetween about 0.1% (w/v) and about 2% (w/v).
 9. The method of claim 8wherein the concentration of said electrolyte in the IL chemoablativepharmaceutical composition is between 0.5 to 1.5% (w/v).
 10. The methodof claim 4 wherein said chemoablative pharmaceutical composition has anosmolality of the composition of greater than about 100 mOsm/kg.
 11. Themethod of claim 8 wherein said electrolyte is sodium chloride.
 12. Themethod of claim 4 wherein said pharmaceutical composition comprises ahydrophilic vehicle.
 13. The method of claim 4 wherein saidpharmaceutical composition has a pH in the range of between about 4 toabout
 10. 14. The method of claim 13 wherein said pharmaceuticalcomposition has a pH in the range of between about 5 to about
 7. 15. Themethod of claim 1 wherein said method is for the treatment of cancersselected from melanoma, breast cancer, primary and metastatic livercancer, prostate cancer and small cell and non small cell lung cancer.16. A method of treatment of cancer comprising administering atherapeutically effective amount of an intralesional chemoablativepharmaceutical composition in combination with a therapeuticallyeffective amount of a systemic targeted anticancer agent.
 17. The methodof claim 16 wherein said systemic targeted anticancer agent is selectedfrom the group consisting of drugs that target protein kinases and thereceptors that activate them, including afatinib (BIBW 2992),bevacizumab, cetuximab, dasatinib, E7080, erlotinib, gefitinib,imatinib, lapatinib, nilotinib, panitumumab, pazopanib, pegaptanib,ranibizumab, sorafenib, sunitinib, trastuzumab and vandetanib;serine/threonine-selective protein kinase inhibitors, including thosetargeting the B-Raf/MEK/ERK pathway, such as vemurafenib (also known asPLX4032, RG7204 or RO5185426), GSK2118436 and GSK1120212; aromataseinhibitors, including aminoglutethimide, anastrozole, exemestane,fadrozole, formestane, letrozole, testolactone and vorozole; estrogenreceptor antagonists, including lasofoxifene, raloxifene, tamoxifen andtoremifene; COX-2 inhibitors, including celecoxib, valdecoxib androfecoxib; angiogenesis blockers, including IFN-α, IL-12, suramin, andthrombospondin (including thrombospondin 1, ABT-510 and ABT-898); andimmune cell therapy, including adoptive T-cell transfer and autologousimmune cell therapy.
 18. The method of claim 16 wherein saidintralesional chemoablative pharmaceutical composition comprises an ILchemoablative agent comprising primarily a halogenated xanthene in anappropriate pharmaceutical composition, including a 0.1% (w/v) or higherconcentration aqueous solution of the halogenated xanthene or mixturesthereof, or a physiologically acceptable salt of the halogenatedxanthene.
 19. The method of claim 18 wherein the halogenated xanthene isrose bengal (4,5,6,7-tetrachloro-2′,4′,5′,7′-tetraiodofluorescein). 20.The method of claim 19 wherein the halogenated xanthene is rose bengaldisodium.
 21. The method of claim 18 wherein the halogenated xanthene isselected from the group consisting of erythrosin B, phloxine B,4,5,6,7-tetrabromo-2′,4′,5′,7′-tetraiodofluorescein,2′,4,5,6,7-pentachloro-4′,5′,7′-triiodofluorescein,4,4′,5,6,7-pentachloro-2′,5′,7′-triiodofluorescein,2′,4,5,6,7,7′-hexachloro-4′,5′-diiodofluorescein,4,4′,5,5′,6,7-hexachloro-2′,7′-diiodofluorescein, 2′,4,5,5′,6,7-hexachloro-4′,7′-diiodofluorescein,4,5,6,7-tetrachloro-2′,4′,5′-triiodofluorescein,4,5,6,7-tetrachloro-2′,4′,7′-triiodofluorescein,4,5,6,7-tetrabromo-2′,4′,5′-triiodofluorescein, and4,5,6,7-tetrabromo-2′,4′,7′-triiodofluorescein.
 22. The method of claim18 wherein said halogenated xanthene has a concentration of about 0.1%(w/v) up to about 20% (w/v), and that the pharmaceutical compositionincludes an electrolyte comprising at least one cation selected from thegroup consisting of sodium, potassium, calcium and magnesium and atleast one anion selected from the group consisting of chloride,phosphate and nitrate, wherein the electrolyte is at a concentration ofbetween about 0.1% (w/v) and about 2% (w/v).
 23. The method of claim 22wherein the concentration of said electrolyte in the IL chemoablativepharmaceutical composition is between 0.5 to 1.5% (w/v).
 24. The methodof claim 18 wherein said chemoablative pharmaceutical composition has anosmolality of the composition of greater than about 100 mOsm/kg.
 25. Themethod of claim 22 wherein said electrolyte is sodium chloride.
 26. Themethod of claim 18 wherein said pharmaceutical composition comprises ahydrophilic vehicle.
 27. The method of claim 18 wherein saidpharmaceutical composition has a pH in the range of between about 4 toabout
 10. 28. The method of claim 27 wherein said pharmaceuticalcomposition has a pH in the range of between about 5 to about
 7. 29. Themethod of claim 16 wherein said method is for the treatment of cancersselected from melanoma, breast cancer, primary and metastatic livercancer, prostate cancer and small cell and non small cell lung cancer.30. A pharmaceutical composition for the treatment of cancer comprising:a therapeutically effective amount of an intralesional chemoablativepharmaceutical composition; and a therapeutically effective amount of asystemic immunomodulatory anticancer agent.
 31. The pharmaceuticalcomposition of claim 30 wherein said systemic immunomodulatoryanticancer agent comprises anti-CTLA-4 antibodies including ipilimumaband tremelimumab.
 32. The pharmaceutical composition of claim 30 whereinsaid systemic immunomodulatory anticancer agent is selected from thegroup consisting of non-specific cytokines, such as interleukin-1,interleukin-2, or interleukin-6 (IL-1, IL-2 or IL-6) and aldesleukin;interferon-alpha or interferon-gamma (IFN-α and IFN-γ), interferonalfa-2b and pegylated interferon (including pegylated interferon alfa-2aand pegylated interferon alfa-2b); granulocyte macrophage colonystimulating factor (GM-CSF, molgramostim or sargramostim); dendriticcell vaccines and other allogeneic or autologous therapeutic cancervaccines, including intralesional vaccines containing an oncolyticherpes virus encoding GM-CSF (OncoVex®) or a plasmid encoding humanleukocyte antigen-B7 and beta-2 microglobulin agent designed to expressallogeneic MHC class I antigens) (Allovectin-7®); and antibodies againstspecific tumor antigens.
 33. The pharmaceutical composition of claim 30wherein said intralesional chemoablative pharmaceutical compositioncomprises an IL chemoablative agent comprising primarily a halogenatedxanthene in an appropriate pharmaceutical composition, including a 0.1%(w/v) or higher concentration aqueous solution of the halogenatedxanthene or mixtures thereof, or a physiologically acceptable salt ofthe halogenated xanthene.
 34. The pharmaceutical composition of claim 33wherein the halogenated xanthene is rose bengal(4,5,6,7-tetrachloro-2′,4′,5′,7′-tetraiodofluorescein).
 35. Thepharmaceutical composition of claim 34 wherein the halogenated xantheneis rose bengal disodium.
 36. The pharmaceutical composition of claim 33wherein the halogenated xanthene is selected from the group consistingof erythrosin B, phloxine B,4,5,6,7-tetrabromo-2′,4′,5′,7′-tetraiodofluorescein,2′,4,5,6,7-pentachloro-4′,5′,7′-triiodofluorescein,4,4′,5,6,7-pentachloro-2′,5′,7′-triiodofluorescein,2′,4,5,6,7,7′-hexachloro-4′,5′-diiodofluorescein,4,4′,5,5′,6,7-hexachloro-2′,7′-diiodofluorescein,2′,4,5,5′,6,7-hexachloro-4′,7′-diiodofluorescein,4,5,6,7-tetrachloro-2′,4′,5′-triiodofluorescein,4,5,6,7-tetrachloro-2′,4′,7′-triiodofluorescein,4,5,6,7-tetrabromo-2′,4′,5′-triiodofluorescein, and4,5,6,7-tetrabromo-2′,4′,7′-triiodofluorescein.
 37. The pharmaceuticalcomposition of claim 33 wherein said halogenated xanthene has aconcentration of about 0.1% (w/v) up to about 20% (w/v), and that thepharmaceutical composition includes an electrolyte comprising at leastone cation selected from the group consisting of sodium, potassium,calcium and magnesium and at least one anion selected from the groupconsisting of chloride, phosphate and nitrate, wherein the electrolyteis at a concentration of between about 0.1% (w/v) and about 2% (w/v).38. The pharmaceutical composition of claim 37 wherein the concentrationof said electrolyte in the IL chemoablative pharmaceutical compositionis between 0.5 to 1.5% (w/v).
 39. The pharmaceutical composition ofclaim 33 wherein said chemoablative pharmaceutical composition has anosmolality of the composition of greater than about 100 mOsm/kg.
 40. Thepharmaceutical composition of claim 37 wherein said electrolyte issodium chloride.
 41. The pharmaceutical composition of claim 33 whereinsaid pharmaceutical composition comprises a hydrophilic vehicle.
 42. Thepharmaceutical composition of claim 33 wherein said pharmaceuticalcomposition has a pH in the range of between about 4 to about
 10. 43.The pharmaceutical composition of claim 42 wherein said pharmaceuticalcomposition has a pH in the range of between about 5 to about
 7. 44. Thepharmaceutical composition of claim 30 wherein said pharmaceuticalcomposition is for the treatment of cancers selected from melanoma,breast cancer, primary and metastatic liver cancer, prostate cancer andsmall cell and non small cell lung cancer.
 45. A pharmaceuticalcomposition for treatment of cancer comprising: a therapeuticallyeffective amount of an intralesional chemoablative pharmaceuticalcomposition; and a therapeutically effective amount of a systemictargeted anticancer agent.
 46. The pharmaceutical composition of claim45 wherein said systemic targeted anticancer agent is selected from thegroup consisting of drugs that target protein kinases and the receptorsthat activate them, including afatinib (BIBW 2992), bevacizumab,cetuximab, dasatinib, E7080, erlotinib, gefitinib, imatinib, lapatinib,nilotinib, panitumumab, pazopanib, pegaptanib, ranibizumab, sorafenib,sunitinib, trastuzumab and vandetanib; serine/threonine-selectiveprotein kinase inhibitors, including those targeting the B-Raf/MEK/ERKpathway, such as vemurafenib (also known as PLX4032, RG7204 orRO5185426), GSK2118436 and GSK1120212; aromatase inhibitors, includingaminoglutethimide, anastrozole, exemestane, fadrozole, formestane,letrozole, testolactone and vorozole; estrogen receptor antagonists,including lasofoxifene, raloxifene, tamoxifen and toremifene; COX-2inhibitors, including celecoxib, valdecoxib and rofecoxib; angiogenesisblockers, including IFN-α, IL-12, suramin, and thrombospondin (includingthrombospondin 1, ABT-510 and ABT-898); and immune cell therapy,including adoptive T-cell transfer and autologous immune cell therapy.47. The pharmaceutical composition of claim 45 wherein saidintralesional chemoablative pharmaceutical composition comprises an ILchemoablative agent comprising primarily a halogenated xanthene in anappropriate pharmaceutical composition, including a 0.1% (w/v) or higherconcentration aqueous solution of the halogenated xanthene or mixturesthereof, or a physiologically acceptable salt of the halogenatedxanthene.
 48. The pharmaceutical composition of claim 47 wherein thehalogenated xanthene is rose bengal(4,5,6,7-tetrachloro-2′,4′,5′,7′-tetraiodofluorescein).
 49. Thepharmaceutical composition of claim 48 wherein the halogenated xantheneis rose bengal disodium.
 50. The pharmaceutical composition of claim 47wherein the halogenated xanthene is selected from the group consistingof erythrosin B, phloxine B,4,5,6,7-tetrabromo-2′,4′,5′,7′-tetraiodofluorescein,2′,4,5,6,7-pentachloro-4′,5′,7′-triiodofluorescein,4,4′,5,6,7-pentachloro-2′,5′,7′-triiodofluorescein,2′,4,5,6,7,7′-hexachloro-4′,5′-diiodofluorescein,4,4′,5,5′,6,7-hexachloro-2′,7′-diiodofluorescein,2′,4,5,5′,6,7-hexachloro-4′,7′-diiodofluorescein,4,5,6,7-tetrachloro-2′,4′,5′-triiodofluorescein,4,5,6,7-tetrachloro-2′,4′,7′-triiodofluorescein,4,5,6,7-tetrabromo-2′,4′,5′-triiodofluorescein, and4,5,6,7-tetrabromo-2′,4′,7′-triiodofluorescein.
 51. The pharmaceuticalcomposition of claim 47 wherein said halogenated xanthene has aconcentration of about 0.1% (w/v) up to about 20% (w/v), and that thepharmaceutical composition includes an electrolyte comprising at leastone cation selected from the group consisting of sodium, potassium,calcium and magnesium and at least one anion selected from the groupconsisting of chloride, phosphate and nitrate, wherein the electrolyteis at a concentration of between about 0.1% (w/v) and about 2% (w/v).52. The pharmaceutical composition of claim 51 wherein the concentrationof said electrolyte in the IL chemoablative pharmaceutical compositionis between 0.5 to 1.5% (w/v).
 53. The pharmaceutical composition ofclaim 47 wherein said chemoablative pharmaceutical composition has anosmolality of the composition of greater than about 100 mOsm/kg.
 54. Thepharmaceutical composition of claim 51 wherein said electrolyte issodium chloride.
 55. The pharmaceutical composition of claim 47 whereinsaid pharmaceutical composition comprises a hydrophilic vehicle.
 56. Thepharmaceutical composition of claim 47 wherein said pharmaceuticalcomposition has a pH in the range of between about 4 to about
 10. 57.The pharmaceutical composition of claim 56 wherein said pharmaceuticalcomposition has a pH in the range of between about 5 to about
 7. 58. Thepharmaceutical composition of claim 45 wherein said pharmaceuticalcomposition is for the treatment of cancers selected from melanoma,breast cancer, primary and metastatic liver cancer, prostate cancer andsmall cell and non small cell lung cancer.